Method of preparing choline salts of bile acids



Patented Mar. 18, 1952 METHOD or PREPARING CHOLINE SALTS or BILE ACIDS Arthur E. Meyer, Flushing, N. Y., assignor to Fellows Medical Manufacturing 00., Inc., New

York, N. Y.

No Drawing. Application August 14, 1948, 1 Serial No. 44,393

4 Claims. (Cl. 260-39'7.1

1 This invention relates to the process of making said choline salts by interaction of alkali salts of bile acids with salts of choline, which process is preferably carried out in an organic solvent and by precipitating the choline bile acid salts with.

other solvents in which they are insoluble.

Choline salts of two bile acids have been prepared before by Gliicksmann. (German Patent 593,258, February 23, 1943.) According to his invention choline sulfate was mixed with a calculated quantity of barium cholate, and the barium sulfate formed was filtered off. The clear filtrate was evaporated to dryness; or choline hydroxide was mixed with an equivalent of 'desoxycholic acid and evaporated to dryness. that the residues so obtained had necessarily the composition and ratio of the ingredients put into the mixture, and there is no proof that one is actually dealing with choline salts. Both methods have their industrial drawbacks because the preparation of choline base is quite cumbersome and the working with the toxic barium compound in the preparation of a pharmaceutical product presents a certain inconvenience although not an absolute objection. The present invention shows a method of preparing the choline salts of certain bile acids avoiding these diificulties and giving products of uniform composition. According to this method, the sodium salts of bile acid, especially of cholic acid, desoxycholic acid or of the natural bile acids, which are a mixture of taurocholie and glycocholic acid, are brought into contact with equivalent quantities of choline chloride. The solvent used for this purpose is preferably ethyl, methyl or any other lower alco-.

hol, although other solvents, such as dioxane, are not excluded. The mixture thus obtained is mixed with an excess of ethyl ether, acetone, or any other ether or ester, commonly used as solvents. This causes the immediat formation of a precipitate that may be of oily consistency or of solid crystalline structure.

On standing at a temperature of about 4 for several days the choline salt in either case assumes its final form. It may be redissolved in alcohol and reprecipitated for further purification although for common use this is not necessary. The sodium chloride formed in the process is sufiiciently soluble in the alcohol-ether mixture so as not to precipitate out with the choline compound. The choline salt, used in this process, does not necessarily have to be the chloride, although this salt is the most commonly available and best suited, but other choline salts such as the sulfate or the dihydrogen It is obvious t citrate may be used as well. The use of the sodium salt of the bile acids is also a matter of in the following: 10

, convenience; the potassium salt or the-salt of any alkaline earth with bile acids may-be used as well, provided the alkali salt formed as a byproduct is removed in the process of precipitation of the bile-acid choline salt with ether.

A description of, the procedures used is given Example 1 -8 grams of desoxycholic acid was dissolved in '100 cc. of ethyl alcohol or methyl alcohol,

'which required moderate heating. Disregarding whether or not the solution was complete, 0.9 gram of sodium hydroxide in 2 cc. of water was added, to form the sodium salt of desoxycholic fjacid. To this was added a solution of 3 grams lated as choline chloride. combined, dissolved in a small quantity of warm choline chloride dissolved in 10 cc. alcohol. A

complete solution was obtained which was poured into 3'volumes of ether. This caused the formation of an oily precipitate. The flask containing the mixture was stoppered and kept over night at 4-5 C. By this time the oil had been transformed into crystals which were quickly filtered off. The ethereal solution gave on addition of /2 vol. more ether after standing at 4-5 C. a

'white precipitate of beautiful crystals. This second yield of crystals was not considerable (about 0.8 gram). Choline determination by the method of Glick (J. Biol. Chem. 1562642 (1944)) showed in both fractions a content of 27.5-28%, calcu- Both fractions were ethyl alcohol, filtered and precipitated by gradual addition of 6 volumes of ethyl ether. The precipitate was white and formed large crystals in the cold. Yield 9 grams. Choline content 275%. as chloride, theory 28.2%.

' The precipitation may also be carried out with acetone or ethyl acetate, but larger quantities of these solvents are needed.

Example 2 4 grams of desoxycholic acid were dissolved in 25 cc. of alcohol.

3 grams of choline dihydrogen citrate was sus- [mended in '75 cc. alcohol and added to the desoxycholic acid solution.

The mixture was heated until complete solution was obtained. 1.35 grams sodium hydroxide in 3 cc. water was added and the mixture was left standing. A few crystals of sodium citrate were formed, which were filtered off and the solution was precipitated with 5 volnmes of ether. After standing in the cold 2 grams of crystals were obtained which were identical with those obtained in experiment 1. Owing to the larger quantities of alcohol necessary for dissolving the material, more ether was necessary for precipitation. The yield was lower than-in experiment 1. Choline dihydrogen citrate is less desirable as a starting material than choline chloride.

Example 3 10 grams cholic acid was dissolved'in12'0 cc. alcohol and 1.2 grams sodium hydroxide in 2 cc. water was added, followed by'3.8 grams choline chloride in 12 cc. alcohol. Precipitation "with ether as described in Example 1 gave crystals .(-9 grams) of choline cholate. (Choline content calculated as chloride 27%. Theory 27.3%.)

Example 4 Iii-"grams cholic acid in '120 cc. "amber-was 'niixed'with 112 grams sodium hydroxidein "8 cc. waterandea grams choline chloride in "12 cc. alcohol. The solution had a pH 9. A'few drops of acetic acid was added'to adjust the reaction t"'7. The crystals precipitated with ether had a choline content of 22.5. They weretreated with 80 cc. of absolute alcohoL'which left a part undissolved. The residue washeated with another- 80 cc. o'f alcohol. Each solution was separately precipitated with 600 .cc. of ether. Both gave crystallizedprecipitates. The first fraction had acholine content of 27.5% calculated'as chloride. lhebsecond fraction .had only 10% choline. The precipitate 'formed at a,.pI-I='7j' contained *accord- .ing to .this experiment some free cholic acid, which remained in-the residue on first extraction with .aalcohol. The soluble part, however, had the theoretical choline content of choline cholate. iThe-choline cholate does not .form a. jelly at any The %-solution.has a ,pH=7.8.

EwampZe'S The mixture .of the sodium salts of tourocholic andglycocholic acids, as is available commercially as"bile salts, was purified by dissolving it in vajsmall volume ofethyl alcohol, adjusting thepH 21to712, filtering and precipitating with an excess of acetone. The ,gummy precipitate was dried auto" I].

733 grams of the purified 'bile salt mixture was dissolved inlOO cc. of alcohol by heating gently and grams of choline chloride was added. on if addition of 4 volumes of acetone a honey like precipitate was obtained. The material was separated 'from the supernatant 'fiuid and heated'at progressively increased temperature up to 110 in diy'air. The semi-solid mass 'containe'd"22% choline calculated as chloride. The theory for choline .taurocholate is 22.6%, for choline glycocholate 24.6%. The theoretical value should be between both figures, however it must be assumed that the choline-bile acid salt contained some moisture that could not be removed; hence the lower choline content. "In repetition of this experiment, using ether as a precipitant a product was obtained that assayed 22.55% choline.

The choline bile salt compounds have a stimiilating'action on the intestine, that difiers from "that'of the bile acids themselves.

I-claim:

51. -A process ofipreparing salts of choline with bile acids by interaction of alkali metal salts of said acids dissolved in a low chain alcohol with *a soluble choline salt and separating the cholinebile-acid salt formed from the alkali metal salt by'*pre'cipitating with a solvent, miscible with alcohol, in which the choline-bile-acid salt is not soluble, selected from the group consisting of 'ethyliether,"acetone and'ethyl acetate.

12. :A1process of preparing choline cholate .by interaction of an alkali metalsalt of cholic acid withfaii'soluble choline salt, both dissolved in a chain-alcohol and separating the choline cholate b'iriprecipitating it with a solvent, miscible with the alcohol, in which the choline cholate is not -soluble, selected from the group consisting of ethyl ether, acetone. and ethyl acetate.

A processiofipreparing choline desoxycholate by intera'ction of anal-kali'metal salt of desoxych'olic acid with a soluble choline salt, both dissolved in alow chain alcoholand'separating the "choline desoxycholate by precipitating it witha s'olvent miscible with the alcohol, in whichthe c'holine desoxyc'holate is insoluble, selected from tlie groupfconsisting of ethyl ether, acetone and ethylacetate.

4. A "process of preparing the choline salt :0 natural bile acids, comprising'a mixture of tau-roc'h'olivand glycocholicacid, by the interaction of 'an alkali 'metaksal't of said'natural bile acids with a sol'ublecholine'salt using a low chainalcohol as-='a- -solvent 'and effecting separation of the reactionpr'oducts by "precipitating the choline salt 'of the n'atural bileacids by theaddition of a solvent-miscible with the alcohol, in which the choline natural bilea'cid salt is insoluble, selected from the group:'consisting'of ethyl ether, 5 acetone and ethyl acetate.

ARTHUR E. MEYER.

REFERENCES CITED v The following references are of 'record in the file ofthis patent:

-FOREIGNlPATENTS Number 25931258 

1. A PROCESS OF PREPARING SALTS OF CHOLINE WITH BILE ACIDS BY INTERACTION OF ALKALI METAL SALTS OF SAID ACIDS DISSOLVED IN A LOW CHAIN ALCOHOL WITH A SOLUBLE CHOLINE SALT AND SEPARATING THE CHOLINEBILE-ACID SALT FORMED FROM THE ALKALI METAL SALT BY PRECIPITATING WITH A SOLVENT, MISCIBLE WITH ALCOHOL, IN WHICH THE CHOLINE-BILE-ACID SALT IS NOT SOLUBLE, SELECTED FROM THE GROUP CONSISTING OF ETHYL ETHER, ACETONE AND ETHYL ACETATE. 